Our long-term goal is to define the biology of the tight junction (TJ) and how its constituent proteins respond to changes in their lipid environment. In this application we focus on occludin's role in signaling to the actin cytoskeleton. Four central questions are addressed using a mouse tracheal epithelial (MTE7b) cell line. 1. What are the consequences of occludin "knock-down" on actin mediated cell functions? Using MTE7b cell clones in which siRNA knocks down occludin by 80-96%, we test for alterations in signaling to the actin cytoskeleton via the GEF-H1/ Rho-GTP/ ROCK1 pathway using a. A novel assay to localize intra- cellular Rho-GTP. b. An apoptotic cell extrusion assay, c. Solute permeability measurements, d. Spinning disc confocal microscopy to examine wound healing. 2. Which occludin domains transduce signals from the plasma membrane to the actin cytoskeleton? MTE7b cells in which endogenous occludin is knocked down by siRNA, are transfected with silently mutated, siRNA-resistant occludin constructs in which specific amino acid sequences in the cytoplasmic and extra-cellular domains are deleted or blocked. The transfected cells are tested for their ability to extrude apoptotic cells, re-organize cortical actin following cholesterol depletion, generate Rho-GTP and alter TJ permeability to large organic cations. 3. Which occludin domains facilitate dendritic cell (DC) migration through TJs while maintaining the TJ barrier? Using MTE7b cells with siRNA-silenced endogenous occludin and transfected with siRNA-resistant occludin deletional mutants, determine the role of specific occludin domains in facilitating the migration through TJs of control DC that express occludin and DC isolated from occludin null mice. 4. What role does occludin and the GEF-H1/ RhoA/ ROCK I signaling pathway play in trans-epithelial DCmigration in response to inhaled antigen? The role of occludin and the GEF-H1/RhoA/ ROCK I pathway is determined in vivo in a model of antigen stimulated DC migration in occludin null mice and their littermate controls. Results of the proposed research will provide new information specifically regarding occludin's recently described signal transducing function and will form the basis for new strategies for regulating the passage of therapeutic agents across the lung epithelium, for facilitating dendritic cell surveillance of the lung and/or for preventing the penetration of allergens across the TJ barrier.